Key intermediates and impurities of the synthesis of Apixaban: Apixaban glycol esters

ABSTRACT

Object of the present invention is an improved process for the preparation of Apixaban, through new intermediates which undergo to a faster amidation reaction. Impurities of Apixaban are also identified and quantified.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 15/038,936,filed May 24, 2016, which is a 371 of PCT/EP2015/053350 filed on Feb.18, 2015, which claims priority to and the benefit of European PatentApplication No. 14189007.9 filed on Oct. 15, 2014, the contents of eachof which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present invention refers to a process for the preparation the activepharmaceutical ingredient named Apixaban through new key intermediates.

BACKGROUND ART

Apixaban is an active pharmaceutical ingredient used as anticoagulantfor the treatment of venous thromboembolic events.

Apixaban has chemical name,1-(4-methoxyphenyl)-7-oxo-6-[4-(2-oxopiperidin-1-yl)phenyl]-4,5-dihydropyrazolo[5,4-c]pyridine-3-carboxamideand has the following chemical formula (I):

Some solvates of Apixaban are known, for example, are known the solvatesof Apixaban with formamide or with dimethylformamide, both havingstoichiometry 1:1.

Apixaban dihydrate, i.e. the hydrate form of Apixaban having twomolecules of water per one of Apixaban is also known.

In literature are disclosed some routes of synthesis of Apixaban, inparticular, in WO2007/0001385 is described in detail the firstindustrial synthesis of Apixaban on multi-Kilos scale.

The PCT application WO2007/0001385 discloses in example 6 a process forthe preparation of Apixaban by amidation reaction on 10 Kg scale of theApixaban ethyl ester according to the following reaction scheme:

According said procedure, using anhydrous ammonia in propylene glycoland performing the reaction for at least 12 hours at 90° C., Apixabanwas obtained with 94.6% of isolated molar yield.

The biggest advantage of the method disclosed in example 6, also incomparison with examples 7 and 9 of WO2007/0001385, is that such amethod provides Apixaban having the polymorphic form named N-1, a solidform which is well characterized in example 9 of the same applicationand which is the thermodynamically stable form of Apixaban.

According to the regulatory information provided by the originator,Apixaban form N-1 is the form currently on the market, so that, with theaim of providing an active pharmaceutical ingredients which providesexactly the same physical-chemical and therapeutical properties of thatthe originator for the generic market, it is important to find a methodfor the preparation of Apixaban which provides the polymorphic form N-1.

In the publication J. Med. Chem., 2007, vol. 50, 22, pag. 5339-5356,Apixaban is prepared from Apixaban ethyl ester with aqueous ammonia at5% in ethylene glycol heating to 120° C. for 4 hours with a molar yieldof 76%, according to the following reaction scheme:

Unfortunately, nothing is said relating to the solid form of Apixabanthus prepared.

The publication Synthetic Communication, 43, pag. 72-79, (2013)discloses a method for the preparation of Apixaban from the intermediateApixaban ethyl ester using 25% aqueous ammonia in methanol at 65° C. for5 hours with 91% molar yield.

Nevertheless such a method, probably because is carried out in Methanolinstead of in a glycol solvent, does not provide Apixaban form N-1,indeed the m.p. of the product is 171-173° C. which is different fromthat of the form N-1 being 235-237° C. Moreover no data relating to thepurity of the product are provided.

In the patent publication WO2013/119328, example 2, the synthesis ofApixaban was carried out from Apixaban ethyl ester using 5% aqueousammonia in propylene glycol at 100° C. overnight. The reaction mixturewas not seeded with the form N-1 so that at the end of the work-up adifferent solid form, named Form I, has been isolated. Apixaban Form Ithus prepared is Apixaban 1,2-propylen glycol hemisolvate.

Considering the above prior art, and our preliminary experimentalresults, the presence of a glycol solvent, such as in example 6 ofWO2007/0001385, seems to promote the preparation of the polymorphic formN-1, while it appears that the presence of an alcohol solvent as inexample 7 and 9 WO2007/0001385 tends to provide the solid form H2-2.

Therefore, to prepare Apixaban form N-1 it appears convenient to isolateApixaban from a glycol solvent.

Nevertheless, although the industrial method for the preparation ofApixaban form N-1 disclosed in WO2007/0001385 already uses a glycolsolvent, such a method has the drawback that it requires long reactiontimes at high temperature, i.e. at least 12 hours at 90° C. or,according to WO2013/119328, 100° C. overnight, or 4 hours at 120° C.(see above J. Med. Chem. (2007)).

DESCRIPTION OF THE FIGURES

FIG. 1 shows the kinetic study of the conversion of Apixaban glycolester to Apixaban in comparison to the conversion of Apixaban ethylester to Apixaban, both conversion carried out under the same amidationconditions.

SUMMARY OF INVENTION

The problem addressed by the present invention is therefore that ofproviding an improved process for the preparation of Apixaban andsolvates or hydrates thereof which avoids long reaction times and/orhigh temperatures.

This problem is solved by a process for the preparation of a Apixabanand salts thereof as outlined in the annexed claims, whose definitionsare integral part of the present description.

Further features and advantages of the process according to theinvention will result from the description hereafter reported ofexamples of realization of the invention, provided as an indication andnot as a limitation of the invention.

DESCRIPTION OF EMBODIMENTS

Object of the present invention is a process for the preparation ofApixaban of formula (I) and solvates or hydrates thereof:

by amidation reaction of the compound of formula (II):

wherein R is chosen from the group comprising a linear or branched C₂-C₆alkyl, —CH₂—CH(OH)—CH₂— and —(R¹O)_(n)R¹— wherein R¹ is a linear orbranched C₂-C₄ alkyl and n is an integer from 1 to 6.

It has been indeed surprisingly found that starting from the compound offormula (II), i.e. from a glycol ester for Apixaban, the amidationreaction to convert it to Apixaban proceed much more faster than usingthe conventional Apixaban C1-C2 alkyl esters.

The effect provided by the compound of formula (II) of the presentinvention is maybe due to the free oxydryl group that in any wayfavourites the substitution of the alkoxy group operated by the ammonia,maybe providing a sort of anchimeric assistance. Alternatively, sucheffect is maybe due to the presence of another oxygen whoseelectronegativity provide an ester which is easily substituted by theammonia.

In other words, the glycol esters of Apixaban of formula (II) areconverted to Apixaban by means of an amidation reaction much more easilyor quickly than the conventional Apixaban esters.

Clear evidences of the effect provided by the process of the inventionare provided in the comparative Table 1 and FIG. 1.

Although in some of the following examples the amidation reactions arecarried out for 6 hours (just to accomplish to an experimental standardprotocol), they were completed well before.

Indeed the amidation reaction of the compound of formula (II) to provideApixaban lasts typically only 3 hours at a temperature comprised between80° C. and 90° C., achieving 99.0% of conversion.

As shown in Table I, under exactly the same conditions the conversion ofApixaban ethyl ester to Apixaban takes at least 6 hours.

According to the industrial process disclosed in WO2007/0001385, saidconversion lasts at least 12 hours at 90° C.

By comparison with the known amidation reaction of Apixaban esters, theprocess of the present invention thus requires shorter reaction times.

As a further advantage of the method of the present invention is thatthe by-product of reaction is a glycol, which can be the solvent mediumof the reaction, thus avoiding the presence of further residual solventsand avoiding the presence of alcohol, e.g. ethanol, as by-product that,according to example 7 and 9 of WO2007/0001385, seems to promote thesolid form H2-2.

The amidation reaction can be performed using anhydrous ammonia, aqueousammonia, ammonium salts such as, for example, ammonium hydroxide,ammonium chloride, ammonium bromide, ammonium sulphate, etc.

The amidation reaction of the process of the present invention can becarried out in an aqueous medium and/or in an organic solvent.

The organic solvent can be an alcohol, glycol, ether, ester, nitrile,hydrocarbon, chlorinated hydrocarbon, etc. and mixtures thereof.

The organic solvent can be an alcohol such as for example, methanol,ethanol, isopropanol, butanol, etc.

The organic solvent can be an ether such as methyl-t-buthyl ether, anester such as ethylacetate or isopropylacetate, a nitrile such asacetonitrile, an hydrocarbon such a toluene or xylene, a chlorinatedhydrocarbon such as chloroform, dichloromethane, chlorobenzene, etc.

The glycol solvents are preferred because they provides Apixaban in thesolid form N-1 and because, using a glycol solvent of formula HO—R—OHwherein R is the same of that of the compound of formula (II), theby-product of the reaction is the same compound of the solvent, thusavoiding an additional residual solvent to be monitored.

The organic solvent can be an a glycol chosen among ethylenglicol,1,2-propilenglycol, 1,3-propilenglycol, diethylenglycol, PEG200,Polypropylenglycol, glycerol.

According to a preferred embodiment of the process of the presentinvention, the preferred solvents are ethylenglicol, 1,2-propylenglycol,1,3-propylenglycol, diethylenglycol.

The glycol solvent of formula HO—R—OH used in the process of theinvention can have the R group with the same meaning of the R group ofthe compound of formula (II), or, alternatively, R can have a differentmeaning. According to a referred embodiment, the glycol solvent offormula HO—R—OH has a R group with the same meaning of the R group ofthe compound of formula (II).

In the compound of formula (II), the R group is chosen from the groupcomprising a linear or branched C₂-C₆ alkyl, —CH₂—CH(OH)—CH₂—,—(R¹O)_(n)R¹— wherein R¹ is a linear or branched C₂-C₄ alkyl an n is aninteger from 1 to 6.

The linear or branched C₂-C₆ alkyl is a group chosen in the groupcomprising —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂(CH₃)—CH₂—, —(CH₂)₄—,—CH(CH₃)CH₂CH₂—, —CH₂CH(CH₃)CH₂, —CH₂CH₂CH(CH₃)—, —(CH₂)₅—, —(CH₂)₆—,etc.

The group —(R¹O)_(n)R¹—, wherein n is an integer from 1 to 6 and whereinR¹ is a linear or branched C₂-C₄ alkyl, is a group chosen in the groupcomprising —(CH₂CH₂O)_(n)CH₂CH₂—, —(CH₂CH₂CH₂O)_(n)CH₂CH₂CH₂—,—(CH(CH₃)CH₂O)_(n)CH(CH₃)CH₂—, —(CH₂CH(CH₃)O)_(n)CH₂CH(CH₃)—,—(CH₂CH₂CH₂CH₂O)_(n)CH₂CH₂CH₂CH₂—, etc.

According to a preferred embodiment, the group —(R¹O)_(n)R¹— is(CH₂CH₂O)_(n)CH₂CH₂—.

According to a preferred embodiment of the present invention, thecompound of formula (II) is a compound isolated, i.e. a compoundisolated from the reaction mixture from which it is prepared. Therefore,the compound of formula (II) is typically in form of a solid or of anisolated oil.

According to a preferred embodiment of the present invention, thecompound of formula (II) is a compound having a purity higher than 80%,measured in HPLC A/A %, for example by using the analytical method ofexample 13.

The process of the present invention is carried out at a temperaturecomprised between 60° C. and 140° C., preferably between 80° C. and 120°C., more preferable between 80° C. and 90° C.

When the amidation reaction is carried out between 80° C. and 90° C.,the reaction is completed (i.e. conversion higher than 99%) in about 3hours.

According to a preferred embodiment, the process of the presentinvention further comprises the step of preparation of the compound offormula (II):

wherein R is chosen from the group comprising a linear or branched C₂-C₆alkyl, —CH₂—CH(OH)—CH₂— and —(R¹O)_(n)R¹— wherein R¹ is a linear orbranched C₂-C₄ alkyl and n is an integer from 1 to 6,

by means of a transesterification reaction of the compound of formula(III):

wherein R² is a linear or branched C₁-C₆ alkyl.

The transesterification of the compound of formula (III) to provide thecompound of formula (II) is thus carried out by reaction of the compoundof formula (III) with a glycol, with a polyglycol or with glycerol.

The transesterification reaction is carried out by reaction of a glycolof formula OH—R—OH where R is chosen from the group comprising a linearor branched C₂-C₆ alkyl, —CH₂—CH(OH)—CH₂— and —(R¹O)_(n)R¹— wherein R¹is a linear or branched C₂-C₄ alkyl and n is an integer from 1 to 6.

Preferred glycols are ethylenglycol, 1,2-propylenglycol,1,3-propylenglycol and diethylenglycol.

Preferred polyglycols are Polyethylenglycol (200) (abbreviated PEG200)and Propylenglicol 200 (PPG200). PEG has the following chemicalstructure HO—(CH₂CH₂O)_(n)CH₂CH₂—OH.

The transesterification reaction can be carried out at a pH comprisedbetween 7.5 and 10.0, preferably at pH comprised between 8.0 and 9.5.

The transesterification reaction can be carried out in presence ofbases, preferably inorganic bases such as, preferably, NaHCO₃ or KH₂PO₄,i.e. bibasic potassium phosphate.

The transesterification reaction is preferably carried out in presenceof bibasic potassium phosphate since it provides the higher and fasterconversions.

The transesterification reaction is carried out at a temperaturecomprised between 60° C. and 120° C., preferably between 70° C. and 80°C., more preferably at about 75° C.

The transesterification reaction is carried out using an excess of thereactant glycol as reaction solvent.

According to an alternative route of synthesis, the compound of formula(II) can be prepared by inner cyclization of the compound of formula(IV):

wherein R is chosen from the group comprising a linear or branched C₂-C₆alkyl, —CH₂—CH(OH)—CH₂— and —(R¹O)_(n)R¹— wherein R¹ is a linear orbranched C₂-C₄ alkyl and n is an integer from 1 to 6.

The compound of formula (IV) can be prepared adapting the known priorart methods used for the preparation of the correspondent esters to thepreparation of said Apixaban glycol ester. Using this syntheticapproach, the compound of formula (II) can be prepared avoiding thepreparation of the previous Apixaban esters of formula (III). Seereaction scheme below.

The cyclization of the compound of formula (IV) to provide the compoundof formula (II) can be carried out in presence of a base such, forexample, t-BuOK.

Moreover, the compound of formula (II) can be prepared according toother synthetic approach which do not necessarily involve thepreparation of the compound of formula (III) or, however, estersintermediates.

In the following reaction scheme is described an example of directsynthesis of the compound of formula (II) by reaction of the glycolester of the hydrazone starting material.

wherein R is chosen from the group comprising a linear or branched C₂-C₆alkyl, —CH₂—CH(OH)—CH₂— and —(R¹O)_(n)R¹— wherein R¹ is a linear orbranched C₂-C₄ alkyl and n is an integer from 1 to 6.

According to a preferred embodiment, in the process of the presentinvention for the preparation of Apixaban and/or in the further step ofpreparation of the compound of formula (II) starting from the compoundof formula (III), the R group in the compound of formula (II) is chosenfrom the group comprising —CH₂CH₂—, —CH₂CH(CH₃)—, CH(CH₃)CH₂—, —(CH₂)₃—and CH₂CH₂OCH₂CH₂—, or according to another embodiment, chosen from thegroup comprising —CH₂CH₂—, —(CH₂)₃— and CH₂CH₂OCH₂CH₂—.

Said compounds of formula (II) can be conveniently prepared bytransesterification of the compound of formula (III), wherein R is forexample ethyl, respectively with ethylenglycol, 1,2-propylenglycol,1,3-propylenglycol and diethylenglycol or, according to anotherembodiment, respectively with ethylenglycol, 1,3-propylenglycol anddiethylenglycol.

The compound of formula (II) prepared from the compound of formula (III)by transesterification with 1,2-propanediol, is a mixture of the twoisomers with ratio 1:2 wherein R is —CH(CH₃)CH₂, named Isomer A, or R is—CH₂CH(CH₃)—, named Isomer B, i.e. having respectively the followingstructures:

It has been observed that the Isomer A (compound (II) withR═—CH(CH₃)CH₂—) has RRT=1,18 according to the analytical methoddescribed in example 13 while the Isomer B (compound (II) withR═—CH₂CH(CH₃)—) has RRT=1,16 according to the same analytical method.

The compound of formula (II) wherein R is —CH₂CH(CH₃)—, i.e. the isomerB, is preferred since this is the main isomer compound prepared bytransesterification reaction of the compound of formula (III) with1,2-propylen glycol.

The compound of formula (II) prepared from the compound of formula (III)by transesterification with ethylenglycol, has instead the only onefollowing structure:

According to a preferred embodiment of the process of the presentinvention, the R substituent of the compound of formula (II) is chosenfrom the group comprising —CH₂CH₂—, —CH₂CH(CH₃)—, —CH(CH₃)CH₂—, —(CH₂)₃—and —CH₂CH₂OCH₂CH₂—.

According to a another preferred embodiment of the of the process of thepresent invention, the R substituent of the compound of formula (II) ischosen from the group comprising —CH₂CH₂—, —(CH₂)₃— and —CH₂CH₂OCH₂CH₂—.

Object of the present invention is thus also the compound of formula(II):

wherein R is chosen from the group comprising a linear or branched C₂-C₆alkyl, —(R¹O)_(n)R¹— wherein R¹ is a linear or branched C₂-C₄ alkyl an nis an integer from 1 to 6, and the group —CH₂—CH(OH)—CH₂—.

According to a preferred embodiment of the compound of formula (II) ofthe present invention, R is chosen from the group comprising —CH₂CH₂—,—CH₂CH(CH₃)—, CH(CH₃)CH₂—, —(CH₂)₃— and —CH₂CH₂OCH₂CH₂—.

According to a another preferred embodiment of the compound of formula(II) of the present invention, R is chosen from the group comprising—CH₂CH₂—, —(CH₂)₃— and —CH₂CH₂OCH₂CH₂—.

The compound of formula (II) of the present invention can be thus usedfor the preparation of Apixaban of formula (I) and solvates or hydratesthereof.

According to an embodiment of the present invention, the process for thepreparation of Apixaban of formula (I) and solvates or hydrates thereof:

comprises the following steps:

a) transesterification reaction of the compound of formula (III):

wherein R² is a linear or branched C₁-C₆ alkyl,

to provide the compound of formula (II):

wherein R is chosen from the group comprising a linear or branched C₂-C₆alkyl, —CH₂—CH(OH)—CH₂— and —(R¹O)_(n)R¹— wherein R¹ is a linear orbranched C₂-C₄ alkyl and n is an integer from 1 to 6,

b) isolating the compound of formula (II),

c) amidation reaction of the compound of formula (II):

to provide Apixaban of formula (I).

According to this above embodiment of the process of the presentinvention, the compound of formula (II) is a useful intermediate for thepreparation of Apixaban which allows to insert a further step in theknown synthesis of Apixaban by direct amidation reaction of Apixabanesters.

Indeed, starting from Apixaban esters of formula (III) and preparing andisolating of the compound of formula (II), allows to increase the puritythe said intermediate of formula (II), thus increasing the purity of thefinal Apixaban.

In other words, performing the process of the invention according saidpreferred embodiment, wherein the intermediate of formula (II) isisolated, by comparison with the known process of direct conversion ofApixaban esters of formula (III) to Apixaban, said process allows theimprove the purity of the final product Apixaban.

The isolation of the compound of formula (II) in the step (b) can becarried out by the known techniques of organic synthesis, includingprecipitation and filtration or centrifugation, or, alternatively,phases separation.

At the end of the step (b) the compound of formula (II) can beoptionally dried, in oven or within the filter drier.

Moreover, isolating the compound of formula (II) can be useful tosatisfy the requirements of Regulatory authorities, which require theprocess being composed by at least three synthetic steps.

According to the just mentioned preferred embodiment of the process ofthe present invention, it is a preferred process that wherein R² in thecompound of formula (III) is ethyl.

According to the just mentioned preferred embodiment of the process ofthe present invention, it is a preferred process that wherein R in thecompound of formula (II) is chosen from the group comprising —CH₂CH₂—,—CH₂CH(CH₃)—, CH(CH₃)CH₂—, —(CH₂)₃— and —CH₂CH₂OCH₂CH₂—.

According to the just mentioned preferred embodiment of the process ofthe present invention, it is another preferred process that wherein R inthe compound of formula (II) is chosen from the group comprising—CH₂CH₂—, —(CH₂)₃— and —CH₂CH₂OCH₂CH₂—.

According to the just mentioned preferred embodiment of the process ofthe present invention, it is a preferred process wherein R² in thecompound of formula (III) is ethyl and wherein R in the compound offormula (II) is chosen from the group comprising —CH₂CH₂—, —CH₂CH(CH₃)—,CH(CH₃)CH₂—, —(CH₂)₃— and —CH₂CH₂OCH₂CH₂—.

According to the just mentioned preferred embodiment of the process ofthe present invention, it is another preferred process wherein R² in thecompound of formula (III) is ethyl and wherein R in the compound offormula (II) is chosen from the group comprising —CH₂CH₂—, —(CH₂)₃— and—CH₂CH₂OCH₂CH₂—.

The conditions to performs the steps (a) and (c) of the preferredembodiment of the invention just mentioned are the same of that alreadydescribed above for the process of the invention, including thepreferred embodiments thereof.

Just like any compound obtained by means of chemical synthesis,Apixaban, and solvates or hydrates thereof, may contain small amounts offoreign compounds referred to as impurities. These impurities may be theraw materials, synthetic intermediaries, reaction by-products,degradation products etc.

The impurities of Apixaban just like those of any other pharmaceuticalactive ingredient or relative drug, referred to as “pharmaceuticalimpurities”, may affect both the efficiency and the safety of a drugwhich, in extreme cases, could even be harmful for the patient. Thepurity of an active ingredient like the Apixaban produced through aproduction process based on subsequent chemical reactions represents acritical factor as regards commercialization. The US Food and DrugAdministration (FDA) and the European Medicinal Agency (EMA) as well asthe relative pharmacopoea require that the impurities be maintainedbelow given limit values.

The product of a chemical reaction is rarely a single compound havingpurity sufficient to meet the regulatory standards. By-products due tosecondary reactions of the reagents used in the reaction can also bepresent in the isolated product. In some steps of the production processof an active ingredient, such as Apixaban, the purity is analysed,generally by means of high performance liquid chromatography (HPLC), gaschromatography (GC) or thin layer chromatography (TLC), for defining ifit is suitable for the subsequent treatment and lastly for use in thepharmaceutical product.

Generally, the impurities are identified spectroscopically, thus achromatographic peak position, such as that of a chromatogram or a spoton a TLC panel, is associated thereto.

Once a peak position has been associated to a particular impurity, theimpurity can be identified in a sample for the relative position thereofin the chromatogram, where the position in the chromatogram is measuredin minutes between the injection of the sample in a column and elutionof the impurity through the detector. The position in the chromatogramis known as the retention time and the ratio between the retention timesis known as the relative retention time.

A man skilled in pharmaceutical art knows that a relatively purecompound may be used as a reference standard. A reference standard issimilar to a reference marker, except for the fact that the latter canbe used not only for detecting the impurities, but also for quantifyingthe amount of impurities present in the sample of active ingredient.

As known to those skilled in the art, the management of processimpurities is considerably improved by understanding the chemicalstructures thereof, the synthetic process and identifying the parametersthat affect the amount of impurities in the final product for example bymeans of DOE. The impurities of Apixaban, including the intermediariesnot entirely reacted, the impurities of the raw materials, the reactionby-products, the degradation products, as well as other products, mayaffect the quality and efficiency of the pharmaceutical form containingApixaban. Thus, there arises the need for a method for defining thelevel of impurities in samples of Apixaban and methods for removing theimpurities or limiting the content thereof or preventing the formationthereof.

As one other aspect of the present invention, during the development ofthe process for the preparation of Apixaban, it has been found that thecompound of formula (II) tends to remain into the product Apixaban, inother words, the compound of formula (II) is both a starting material orintermediate for the synthesis of Apixaban according to the method ofthe present invention and is also an impurity of Apixaban.

In order to reduce the amount of an impurity in an active ingredient itis necessary to detect the presence thereof using appropriate analyticalmethods, it is convenient to identify it, quantify it and onlyafterwards one can provide a method of synthesis capable of preventingthe formation and/or provide for the removal thereof. However, thisessentially requires providing the reference standard or referencemarker of this impurity. For such purpose the compound of formula (II)can be conveniently prepared by means of the method described above.

The compound of formula (II) of the present invention can be thus usedas reference marker or reference standard for the identification and/orquantification of said compound of formula (II) in Apixaban and solvatesor hydrates thereof.

The compound of formula (II) can be indeed used according to thefollowing analytical methods the identification and/or quantification ofsaid compound of formula (II) in Apixaban and solvates or hydratesthereof.

A method for detecting or identifying the compound of formula (II) inApixaban or a solvate or hydrate thereof comprises:

a) adding a known amount of compound of formula (II) to the Apixabansample or a solvate or hydrate thereof,

b) carrying out HPLC analysis of the Apixaban sample or a solvate orhydrate thereof of step a),

c) detecting the HPLC peak of the compound of formula (I);

or,

a1) analysing the compound of formula (II) by means of HPLC,

b1) analysing the Apixaban sample or a solvate or hydrate thereof bymeans of HPLC,

c1) detecting the HPLC peak of the compound of formula (II) by comparingthe retention times or relative retention times.

Substantially using the method above, it allowed identifying the peak inthe chromatogram of Apixaban sample or a solvate or hydrate thereofregarding the impurity compound of formula (II). The analysis may be ofthe HPLC type.

Besides the identification of the impurity peak in Apixaban or a solvateor hydrate, a method for the quantification of the compound of formula(II) in Apixaban or a solvate or hydrate thereof comprises:

i) measuring the peak area corresponding to the compound of formula (II)in a Apixaban sample or a solvate or hydrate thereof having an unknownamount of compound of formula (II) by means of HPLC;

ii) measuring the peak area corresponding to a reference standardcontaining a known amount of compound of formula (II) by means of HPLC;

iii) defining the amount of compound of formula (II) in Apixaban or asolvate or hydrate thereof comparing the area measured in step a) withthat measured in step ii).

It is thus clear that the compound of formula (II) may be used as areference marker or reference standard respectively for theidentification and/or the quantification of the same in Apixaban or asolvate or hydrate thereof.

In particular, it has been observed that the impurity of Apixabanproduced according to the process of the present invention and havingRRT=1.16 with the analytical method reported in example 13 is the IsomerB of the following structures, while the impurity having RRT=1.18 hasthe is the Isomer A of the following structures:

As an aspect related to the solid form of Apixaban produced by theprocess of the present invention, a study directed to the preparation ofApixaban solid form N-1 was carried out.

Repeating the experiment of example 6 of WO2007/0001385 but withoutadding the seed of form N-1, it has been obtained, prepared andcharacterized Apixaban 1,2-Proprylen glycol hemisolvate of formula (V):

Apixaban 1,2-propylene glycol solvate having stoichiometry (2:1) offormula (V) is a white solid.

After that, a study directed to find a method to obtain N-1 form fromApixaban 1,2-proprylen glycol hemisolvate of formula (V) was carriedout.

A screening using Apixaban 1,2-propylene glycol hemisolvate (abbreviatedAPX•PG) as starting material was performed using form N-1 as a seed inall the cases. The experiments were performed with ICH guideline class 3solvents (except MeOH, ICH guideline class 2 with a residual solventpermitted of 3000 ppm). The low solubility of APX•PG with commonindustrial solvents limited the use of the crystallization.

The successful experiments that provided Apixaban form N-1 from APX•PGare collected in Table 1.

TABLE 1 Methods of preparation of Apixaban form N-1 from APX•PG. # scalemethod solvent T° time Yield 1 250 mg Crystallization MeOH Reflux 4hours 76% (36 v.) →rT° 2 250 mg Crystallization EtOH Reflux 4 hours 83%(60 v.) →rT° 3 100 mg Slurrying MeOH rT° overnight 87% (10 v.) 4 up to 1g Slurrying EtOH rT° 4 hours 86% (10 v.) 5 up to 1 g Slurrying IPA 50°C. overnight 89% (10 v.) 6 500 mg Slurrying EtOH/ rT° 6 hours 90%heptane (10 v.) 7  50 mg Slurrying EtOAc rT° overnight 93% (10 v.)

Slurrying seems to be the best procedure to prepare form N-1 because ahigh amount of solvent was required in crystallizations (36-60 volumes).

Due to their lower boiling points and the good industry acceptance, EtOHand IPA were selected as solvents to perform a scale-up of the slurryingexperiments to 1 g. The transformation was monitored by XRPD:

-   -   In EtOH the transformation was finished after 1 hour at room        temperature.    -   In IPA the transformation is much slower: after 5 h at 50° C.,        the conversion was not complete, but finished after one night.

Propylene glycol was not detected in Apixaban form N-1 by ¹H-NMR.Unfortunately, residual solvent was detected by ¹H-NMR in bothexperiments (approx. 0.8 wt % of EtOH and 0.7 wt % of IPA). The NMRanalysis indicated also 0.7 wt % of residual solvent when EtOAc wasused.

¹H-NMR analyses of form N-1 obtained in EtOH by crystallization andslurrying indicated that the amount of residual solvent is lower in thecase of the crystallization (0.4 wt % instead of 0.8 wt %). The methodof preparation seems to have some effect on the final amount of residualsolvent (perhaps due to the different particle sizes or kind ofaggregates).

Typically, the Apixaban form N-1 prepared according to the process ofthe invention has a water content comprised between 0.05% and 0.1%.

EXPERIMENTAL SECTION

The starting material Apixaban ethyl ester can be prepared according toExample 5 of WO2007/001385.

Example 1: Preparation of Apixaban 1,2-Propylen Glycol Hemisolvate ofFormula (V) from Apixaban Ethyl Ester

In an autoclave inerted by nitrogen Apixaban ethyl ester (15 g, 1.0 eq)and propylene glycol (1,2-propan diol, 135 mL) were charged and thevessel was pressurized with ammonia at p=4 bar and T=80/85° C. for 6 h.The mixture was then transferred in a round bottom flask, cooled to45/50° C. and diluted with water (85 mL). After stirring at T=45/50° C.for additional 2 h, the suspension was cooled to 20/25° C. for 10 h andfiltered. The wet cake was washed with water (2×30 mL). The solid wasdried under vacuum at T=75° C. for 8 h affording Apixaban1,2-propylenglycol hemisolvate of formula (V) (13.3 g, 0.86 eq). m.p.195° C. ¹H-NMR (400 MHz, CDCl₃, ppm), δ: 7.49 (d, J=9 Hz, 2H), 7.35 (d,J=8 Hz, 2H), 7.28 (d, J=8 Hz, 2H), 6.95 (d, J=9 Hz, 2H), 6.91 (s, 1H),5.91 (s, 1H), 4.13 (t, J=8 Hz, 2H), 3.84 (bs, 3H+0.5H CH propyleneglycol), 3.62 (bm, 2H+0.5H OH propylen glycol), 3.39 (bm, J=8 Hz,2H+0.5H OH propylen glycol), 2.57 (bs, 2H+1H CH₂ propylen glycol), 1.95(bs, 4H), 1.14 (d, J=6.4 Hz, 1.5H CH₃ propylen glycol). 13C NMR and DEPT135 NMR (100 MHz, CDCl₃, ppm), δ: 170.3 (C), 163.8 (C), 159.9 (C), 157.4(C), 141.4 (C), 140.7 (C), 140.0 (C), 133.4 (C), 132.5 (C), 126.8 (CH),126.2 (CH), 126.5 (CH), 125.9 (CH), 113.8 (CH), 68.3 (CH), 68.1 (CH₂),55.6 (CH₃), 51.6 (CH₂), 51.2 (CH₂), 32.8 (CH₂), 23.5 (CH₂), 21.4 (CH₂),21.3 (CH₂), 18.8 (CH₃). ESI-MS m/z=460 ([M+H]⁺). IR (ATR, cm⁻¹): 3447,3145, 2940, 2860, 1687, 1631, 1543, 1512, 1465, 1441, 1401, 1380, 1350,1326, 1297, 1243, 1170, 1144, 1111, 1027, 1016, 982, 945, 831, 812, 761,705. X-RPD (2θ°): 6.6°, 7.6°, 8.1°, 9.9°, 11.7°, 12.7°, 13.7°, 14.5°,15.1°, 15.6°, 16.3°, 16.9°, 17.2°, 17.9°, 18.2°, 19.5°, 20.0°, 20.5°,20.8°, 21.4°, 22.8°, 23.8°, 24.8°, 25.5°, 29.0°, 31.2°, 33.0°.

The reworking of example 6 of WO2007/001385, carried out many times butwithout adding the seeds of Apixaban form N-1, always provided Apixaban1,2-propylen glycol hemisolvate of formula (V).

This is in agreement with the teachings of the patent publicationWO2013/119328 wherein Apixaban form I, i.e. Apixaban 1,2-propylen glycolhemisolvate was obtained without the seeding with form N-1.

Example 2: Preparation of Apixaban Form N-1 from Apixaban 1,2-PropylenGlycol Hemisolvate—without Seeding of Form N-1

In a round bottom flask were charged Apixaban 1,2-propylene glycolhemisolvate (10 g, 1.0 eq) and ethanol (400 mL) and the mixture washeated to reflux for 4 hours. The suspension was slowly cooled to 20/25°C. and stirred at this temperature for 8 h, then filtered washing withethanol (2×20 mL). The wet solid was dried under vacuum at 75° C. for 8h affording 8.1 g of Apixaban N-1 form (0.87 eq). mp 237° C. ¹H-NMR (400MHz, DMSO-d₆, ppm), δ: 7.74 (s, 1H), 7.53 (d, J=12 Hz, 2H), 7.47 (s,1H), 7.37 (d, J=8 Hz, 2H), 7.30 (d, J=12 Hz, 2H), 7.02 (d, J=8 Hz, 2H),4.07 (t, J=8 Hz, 2H), 3.82 (s, 3H), 3.61 (t, J=4 Hz, 2H), 3.23 (t, J=8Hz, 2H), 2.41 (t, J=4 Hz, 2H), 1.87 (m, 4H). ¹³C-NMR and DEPT 135 NMR(100 MHz, DMSO-d₆, ppm), δ: 169.3 (C), 163.7 (C), 159.6 (C), 157.1 (C),142.0 (C), 141.9 (C), 140.3 (C), 133.5 (C), 133.1 (C), 127.3 (C), 126.8(CH), 126.5 (CH), 125.7 (CH), 113.9 (CH), 56.0 (CH₃), 51.3 (CH₂), 33.1(CH₂), 23.5 (CH₂), 21.5 (CH₂), 21.4 (CH₂). ESI-MS m/z=460 ([M+H]⁺).

Example 3: Characterization of Apixaban Form N-1

Apixaban form N-1 obtained by crystallization in EtOH was characterizedby several techniques.

FT-IR

FTIR spectrum was recorded using a Thermo Nicolet Nexus 870 FT-IR,equipped with a beamsplitter KBr system, a 35 mW He—Ne laser as theexcitation source and a DTGS KBr detector. The spectrum was acquired in32 scans at a resolution of 4 cm⁻¹.

IR (KBr): v=3483 (m), 3311 (m), 2909 (m), 2866 (W), 1683 (s), 1630 (s),1595 (s), 1519 (m), 1295 (m), 1256 (m), 975 (m), 848 (s), 813 (m), 668(m), 467 (m) cm⁻¹ (see FIG. 2).

DSC

DSC analysis was recorded with a Mettler DSC822^(e). A sample of 1.6770mg was weighed into a 40 μL aluminium crucible with a pinhole lid andwas heated, under nitrogen (50 mL/min), at 10° C./min from 30 to 300° C.

Form N-1 is characterized by an endothermic sharp peak corresponding tothe melting point with an onset at 235.68° C. (fusion enthalpy−106.66J/g), measured by DSC analysis (10° C./min).

TGA

Thermogravimetric analysis was recorded in a thermogravimetric analyzerMettler TGA/SDTA851^(e). A sample of 4.2206 mg was weighed into a 70 μLalumina crucible with a pinhole lid and was heated at 10° C./min from 30to 400° C., under nitrogen (50 mL/min).

The TG analysis of Form N1 shows a 0.23% weight loss before the meltingpoint (between 130° C. and 230° C.). This loss of weight could come fromthe elimination of EtOH traces.

X-RPD

XRPD analysis was performed using a PANalytical X'Pert diffractometerwith Cu Kα radiation in Bragg-Brentano geometry. The system is equippedwith a monodimensional, real time multiple strip detector. Thediffractogram was recorded from 3° to 40° (2θ) at a scan rate of 17.6°per minute (see FIG. 5). List of selected peaks (only peaks withrelative intensity greater than or equal to 1% are indicated):

Pos. Rel. Int. [°2Th.] [%] 8.4 9 10.0 4 10.5 5 11.1 5 12.3 8 12.8 4113.9 58 15.1 2 16.2 14 16.9 100 18.4 30 18.8 14 19.6 8 21.1 11 21.5 1222.0 16 22.2 29 23.6 2 24.0 4 24.7 8 25.3 4 26.2 1 26.9 8 27.7 5 28.0 328.6 4 29.2 6 29.9 5 30.6 3 31.9 1 32.6 5 35.1 3

In the U.S. Pat. No. 7,396,932B2, form N-1 was described by SCXR and ¹³CSSNMR. Using the data of SCXR (unit cell, symmetry and atom positions),XRPD was simulated using the Mercury program. Comparison of thissimulated XRPD with the experimental XRPD obtained in example 2confirmed the formation Apixaban N-1 form.

Karl Fischer

Karl Fischer analyses were recorded with a Metrohm 787 KF Trinito. Theproduct was dissolved in MeOH. Two samples were analyzed using thefollowing reactants: Hydranal-Composite 5 (Riedel de Haën Ref. 34805),Hydranal Methanol Rapid (Riedel de Haën Ref. 37817) and Hydranal WaterStandard 1.0 (Riedel de Haën Ref. 34828 used to calculate the factor).

The water content of form N-1 prepared in example 2 is 0.9%.

Example 4: Preparation of Apixaban Form N-1 from Apixaban 1,2-ProprylenGlycol Hemisolvate on Larger Scale—with Seeding of Form N-1

To a three-necked round-bottomed flask equipped with a thermometer andmechanical stirrer was added Apixaban 1,2-Propylene glycol hemisolvateof formula (V) (85.1 g; 171 mmol), as prepared in Example 1, and amixture of EtOH/water (2:1) (850 mL, 10 vol.). the resulting suspensionwas seeded with form N-1 (as prepared in example 2) and it was heated at50° C. The mixture was maintained at 50° c. for 2.5 hours and then itwas cooled down to room temperature. The slurry was stirred at roomtemperature for 2-3 hours. The solid was filtered with a sintered funnel(porosity 2-very good filtration), washed with EtOH: water (2:1) (170mL, 2 vol.), with water (170 mL, 2 vol.) and dried under vacuum at 50°C. overnight. Apixaban form N-1 was obtained as off-white powder (65.4g, 83% yield). ¹H-NMR analysis shows that the product contains 0.13% ofresidual Ethanol. K. F. 0.1%. The chemical purity was determinate byHPLC: 99.4%. The starting Apixaban 1,2-Propylene glycol hemisolvate hada purity of 98.3%.

Repeating the above procedure starting from Apixaban 1,2-Propyleneglycol hemisolvate with a purity of 95.2%, Apixaban form N-1 wasprepared having a purity of 99.5%.

Example 5: Synthesis of the Compound of Formula (II) in which R is—CH₂CH₂—

A mixture of Apixaban ethyl ester (10.0 g, 1.0 eq), bibasic potassiumphosphate (K₂HPO₄, 17.8 g, 5.0 eq) and ethylenglycol (1,2-ethan diol, 70mL) was heated to T=75° C. for 10 h and then cooled to room temperature.Water (70 mL) and dichloromethane (70 mL) were charged and the resultingbiphasic solution was stirred at room temperature for 10 min. Once cutthe phases, the organic layer was treated with molecular sieves toremove residual water and then concentrated to residue at reducedpressure. The resulting solid was employed without further purification(9.0 g, 0.87 eq). ¹H-NMR (400 MHz, DMSO-d₆, ppm), δ: 7.52 (d, J=12 Hz,2H), 7.37 (d, J=8 Hz, 2H), 7.30 (d, J=8 Hz, 2H), 7.03 (d, J=12 Hz, 2H),4.98 (t, J=4 Hz, 1H), 4.35 (t, J=4 Hz, 2H), 4.09 (t, J=4 Hz, 2H), 3.82(s, 3H), 3.74 (dd, J₁=4 Hz, J₂=4 Hz, 2H), 3.60 (t, J=4 Hz, 2H), 3.24 (t,J=4 Hz, 2H), 2.40 (t, J=4 Hz, 2H), 1.85 (m, 4H). ¹³C-NMR and DEPT 135NMR (100 MHz, DMSO-d₆, ppm), δ: 169.4 (C), 162.0 (C), 159.8 (C), 156.9(C), 141.9 (C), 140.2 (C), 139.0 (C), 133.5 (C), 132.9 (C), 127.4 (CH),127.2 (CH), 126.8 (CH), 126.5 (CH), 114.0 (CH), 66.8 (CH₂), 59.5 (CH₂),56.0 (CH₃), 51.3 (CH₂), 51.2 (CH₂), 33.1 (CH₂), 23.5 (CH₂), 21.6 (CH₂),21.4 (CH₂). ESI-MS m/z=505 ([M+H]⁺).

Example 6: Synthesis of the Compound of Formula (II) in which R is—CH(CH₃)₂CH₂— and —CH₂CH(CH₃)—

A mixture of Apixaban ethyl ester (30.0 g, 1.0 eq.), bibasic potassiumphosphate (K₂HPO₄, 53.4 g, 5.0 eq.) and propylenglycol (1,2-propan diol,210 mL) was heated to T=75° C. for 10 h and then cooled to roomtemperature. Water (210 mL) and dichloromethane (210 mL) were chargedand the resulting biphasic solution was stirred at room temperature for10 min. Once cut the phases, the organic layer was treated withmolecular sieves to remove residual water and then concentrated toresidue at reduced pressure. The resulting solid is a 1:2 mixture of thetwo isomers (called isomer A and isomer B respectively in the ¹H-NMRcharacterization) was employed without further purification (25.2 g,0.79 eq). ¹H-NMR (400 MHz, DMSO-d₆, ppm), δ: 7.52 (m, 3H (2H isomer Aand 2H isomer B)), 7.37 (m, 3H (2H isomer A and 2H isomer B)), 7.30 (m,3H (2H isomer A and 2H isomer B)), 7.03 (m, 3H (2H isomer A and 2Hisomer B)), 5.12 (m, 0.5H (1H isomer A), 4.96 (m, 1.5H (1H isomer A and1H isomer B)), 4.20 (m, 2H (2H isomer B)), 4.11 (m, 3H (2H isomer A and2H isomer B)), 3.98 (m, 1H (1H isomer B)), 3.83 (m, 4.5H (3H isomer Aand 3H isomer B)), 3.61 (m, 3H (2H isomer A and 2H isomer B)), 3.26 (m,3H (2H isomer A and 2H isomer B)), 1.88 (m, 6H (4H isomer A and 4Hisomer B)), 1.29 (d, J=4 Hz, 1.5H (3H isomer A)), 1.17 (d, J=4 Hz, 3H(3H isomer B)). ¹³C-NMR (100 MHz, DMSO-d₆, ppm), δ: 169.4, 161.8, 161.6,159.8, 156.9, 141.9, 140.2, 139.3, 139.0, 133.5, 133.0, 127.3, 127.2,126.8, 126.5, 114.0, 72.7, 69.9, 64.5, 64.1, 56.0, 51.3, 51.2, 33.1,23.5, 21.7, 21.4, 20.5, 16.9 (overlap of some of the two isomers signalswas observed). ESI-MS m/z=519 ([M+H]⁺).

IR (ATR, cm⁻¹): 3329, 2934, 2839, 1708, 1673, 1627, 1592, 1511, 1438,1403, 1372, 1325, 1301, 1252, 1172, 1144, 1054, 1021, 988, 949, 832,788, 699. X-RPD (2θ°): 6.7°, 8.2°, 8.5°, 8.9°, 10.5°, 11.1°, 11.6°,12.1°, 13.0°, 15.3°, 15.9°, 16.8°, 17.2°, 17.9°, 19.3°, 20.1°, 20.4°,21.3°, 22.8°, 23.2°, 23.8°, 24.5°, 25.4°, 27.9°, 30.3°.

Example 7: Synthesis of the Compound of Formula (II) in which R is—CH₂CH₂OCH₂CH₂—

A mixture of Apixaban ethyl ester (10.0 g, 1.0 eq), bibasic potassiumphosphate (K₂HPO₄, 17.8 g, 5.0 eq) and diethylenglycol (70 mL) washeated to T=75° C. for 10 h and then cooled to room temperature. Water(70 mL) and dichloromethane (70 mL) were charged and the resultingbiphasic solution was stirred at room temperature for 10 min. Once cutthe phases, the organic layer was treated with molecular sieves toremove residual water and then concentrated to residue at reducedpressure. The resulting solid was employed without further purification(10.5 g, 0.93 eq). ¹H-NMR (400 MHz, DMSO-d₆, ppm), δ: 7.53 (d, J=8 Hz,2H), 7.37 (d, J=8 Hz, 2H), 7.30 (d, J=8 Hz, 2H), 7.03 (d, J=8 Hz, 2H),4.65 (t, J=4 Hz, 1H), 4.45 (m, 2H), 4.10 (d, J=4 Hz, 2H), 3.83 (s, 3H),3.77 (m, 2H), 3.53 (m, 4H), 7.53 (d, J=8 Hz, 2H), 3.24 (d, J=8 Hz, 2H),2.41 (m, 2H), 1.86 (m, 4H). ¹³C-NMR and DEPT 135 NMR (100 MHz, DMSO-d₆,ppm), δ: 169.4 (C), 161.8 (C), 159.8 (C), 156.9 (C), 141.9 (C), 140.2(C), 138.8 (C), 133.5 (C), 132.9 (C), 127.5 (C), 127.2 (CH), 126.8 (CH),126.5 (CH), 114.0 (CH), 72.8 (CH₂), 68.7 (CH₂), 64.3 (CH₂), 60.7 (CH₂),56.0 (CH₃), 51.3 (CH₂), 51.2 (CH₂), 33.1 (CH₂), 23.5 (CH₂), 21.6 (CH₂),21.4 (CH₂). ESI-MS m/z=549 ([M+H]⁺). KF=0.06%.

Example 8: Synthesis of Apixaban Form N-1 from the Compound of Formula(II) in which R is —CH₂CH₂—

In an autoclave inerted by nitrogen compound of Example 5 (compound offormula (II) in which R is —CH₂CH₂—, 8.0 g, 1.0 eq) and propylene glycol(1,2-propandiol, 80 mL) were charged and the vessel was pressurized withammonia at p=4 bar and T=80/85° C. for 6 h. The mixture was thentransferred in a round bottom flask, heated to dissolution and dilutedwith water (16 mL). After stirring at T=95/100° C. for additional 2 h,more water was added (48 mL) and the solution was seeded with ApixabanN-1 form (as prepared in Example 2 or 4). The sus-pension was stirredfor 2 h at T=95/100° C., cooled to room temperature and diluted withethanol (16 mL). After 3 h stirring at T=20/25° C. the slurry wasfiltered and the wet cake was washed with water (2×8 mL). The solid wasdried under vacuum at T=65° C. for 8 h affording Apixaban N-1 form (6.7g, 0.92 eq). m.p. 237° C. ¹H-NMR (400 MHz, DMSO-d₆, ppm), δ: 7.74 (s,1H), 7.53 (d, J=12 Hz, 2H), 7.47 (s, 1H), 7.37 (d, J=8 Hz, 2H), 7.30 (d,J=12 Hz, 2H), 7.02 (d, J=8 Hz, 2H), 4.07 (t, J=8 Hz, 2H), 3.82 (s, 3H),3.61 (t, J=4 Hz, 2H), 3.23 (t, J=8 Hz, 2H), 2.41 (t, J=4 Hz, 2H), 1.87(m, 4H). ¹³C-NMR and DEPT 135 NMR (100 MHz, DMSO-d₆, ppm), δ: 169.3 (C),163.7 (C), 159.6 (C), 157.1 (C), 142.0 (C), 141.9 (C), 140.3 (C), 133.5(C), 133.1 (C), 127.3 (C), 126.8 (CH), 126.5 (CH), 125.7 (CH), 113.9(CH), 56.0 (CH₃), 51.3 (CH₂), 33.1 (CH₂), 23.5 (CH₂), 21.5 (CH₂), 21.4(CH₂). ESI-MS m/z=460 ([M+H]⁺). KF=0.08%.

Example 9: Synthesis of Apixaban Form N-1 from the Compound of Formula(II) in which R is —CH(CH₃)₂CH₂— and —CH₂CH(CH₃)—

In an autoclave inerted by nitrogen compound of Example 6 (compound offormula (II) in which R is —CH(CH₃)₂CH₂— and —CH₂CH(CH₃)—, 11 g, 1.0 eq)and propylene glycol (1,2-propandiol, 100 mL) were charged and thevessel was pressurized with ammonia at p=4 bar and T=80/85° C. for 6 h.The mixture was then transferred in a round bottom flask, heated todissolution and diluted with water (20 mL). After stirring at T=95/100°C. for additional 2 h, more water was added (60 mL) and the solution wasseeded with Apixaban N-1 form. The suspension was stirred for 2 h atT=95/100° C., cooled to room temperature and diluted with ethanol (20mL). After 3 h stirring at T=20/25° C. the slurry was filtered and thewet cake was washed with water (2×10 mL). The solid was dried undervacuum at T=65° C. for 8 h affording Apixaban N-1 form (8.6 g, 0.88 eq).m.p. 237° C. ¹H-NMR (400 MHz, DMSO-d₆, ppm), δ: 7.74 (s, 1H), 7.53 (d,J=12 Hz, 2H), 7.47 (s, 1H), 7.37 (d, J=8 Hz, 2H), 7.30 (d, J=12 Hz, 2H),7.02 (d, J=8 Hz, 2H), 4.07 (t, J=8 Hz, 2H), 3.82 (s, 3H), 3.61 (t, J=4Hz, 2H), 3.23 (t, J=8 Hz, 2H), 2.41 (t, J=4 Hz, 2H), 1.87 (m, 4H).¹³C-NMR and DEPT 135 NMR (100 MHz, DMSO-d₆, ppm), δ: 169.3 (C), 163.7(C), 159.6 (C), 157.1 (C), 142.0 (C), 141.9 (C), 140.3 (C), 133.5 (C),133.1 (C), 127.3 (C), 126.8 (CH), 126.5 (CH), 125.7 (CH), 113.9 (CH),56.0 (CH₃), 51.3 (CH₂), 33.1 (CH₂), 23.5 (CH₂), 21.5 (CH₂), 21.4 (CH₂).ESI-MS m/z=460 ([M+H]⁺). KF=0.05%.

Example 10: Synthesis of Apixaban Form N-1 from the Compound of Formula(II) in which R is —CH₂CH₂OCH₂CH₂—

In an autoclave inerted by nitrogen compound of Example 7 (compound offormula (II) in which R is —CH₂CH₂OCH₂CH₂—, 9.0 g, 1.0 eq) and propyleneglycol (1,2-propandiol, 105 mL) were charged and the vessel waspressurized with ammonia at p=4 bar and T=80/85° C. for 6 h. The mixturewas then transferred in a round bottom flask, heated to dissolution anddiluted with water (20 mL). After stirring at T=95/100° C. foradditional 2 h, more water was added (60 mL) and the solution was seededwith Apixa-ban N-1 form. The suspension was stirred for 2 h at T=95/100°C., cooled to room temperature and diluted with ethanol (20 mL). After 3h stirring at T=20/25° C. the slurry was filtered and the wet cake waswashed with water (2×10 mL). The solid was dried under vacuum at T=65°C. for 8 h affording Apixaban N-1 form (6.5 g, 0.86 eq). mp 237° C.¹H-NMR (400 MHz, DMSO-d₆, ppm), δ: 7.74 (s, 1H), 7.53 (d, J=12 Hz, 2H),7.47 (s, 1H), 7.37 (d, J=8 Hz, 2H), 7.30 (d, J=12 Hz, 2H), 7.02 (d, J=8Hz, 2H), 4.07 (t, J=8 Hz, 2H), 3.82 (s, 3H), 3.61 (t, J=4 Hz, 2H), 3.23(t, J=8 Hz, 2H), 2.41 (t, J=4 Hz, 2H), 1.87 (m, 4H). ¹³C NMR and DEPT135 NMR (100 MHz, DMSO-d₆, ppm), δ: 169.3 (C), 163.7 (C), 159.6 (C),157.1 (C), 142.0 (C), 141.9 (C), 140.3 (C), 133.5 (C), 133.1 (C), 127.3(C), 126.8 (CH), 126.5 (CH), 125.7 (CH), 113.9 (CH), 56.0 (CH₃), 51.3(CH₂), 33.1 (CH₂), 23.5 (CH₂), 21.5 (CH₂), 21.4 (CH₂). ESI-MS m/z=460([M+H]⁺). KF=0.06%.

Example 11: Synthesis of Apixaban Form N-1—from the Compound of Formula(III) in which R is —CH₂CH₃ (Example 6 of WO2007/0001385)—ComparativeExample

In an autoclave inerted by nitrogen Apixaban ethyl ester (65 g, 1.0 eq)and propylene glycol (1,2-propan diol, 455 mL) were charged and thevessel was pressurized with ammonia at p=4 bar and T=80/85° C. for 6 h.The mixture was then transferred in a round bottom flask washing theautoclave with propylene glycol (65 mL), heated to dissolution anddiluted with water (130 mL). After stirring at T=95/100° C. foradditional 2 h, more water was added (390 mL) and the solution wasseeded with Apixaban N-1 form. The suspension was stirred for 2 h atT=95/100° C., cooled to room temperature and diluted with ethanol (130mL). After 3 h stirring at T=20/25° C. the slurry was filtered and thewet cake was washed with water (2×130 mL). The solid was dried undervacuum at T=65° C. for 8 h affording Apixaban N-1 form (56.0 g, 0.917eq). m.p. 237° C. ¹H-NMR (400 MHz, DMSO-d₆, ppm), δ: 7.74 (s, 1H), 7.53(d, J=12 Hz, 2H), 7.47 (s, 1H), 7.37 (d, J=8 Hz, 2H), 7.30 (d, J=12 Hz,2H), 7.02 (d, J=8 Hz, 2H), 4.07 (t, J=8 Hz, 2H), 3.82 (s, 3H), 3.61 (t,J=4 Hz, 2H), 3.23 (t, J=8 Hz, 2H), 2.41 (t, J=4 Hz, 2H), 1.87 (m, 4H).¹³C-NMR and DEPT 135 NMR (100 MHz, DMSO-d₆, ppm), δ: 169.3 (C), 163.7(C), 159.6 (C), 157.1 (C), 142.0 (C), 141.9 (C), 140.3 (C), 133.5 (C),133.1 (C), 127.3 (C), 126.8 (CH), 126.5 (CH), 125.7 (CH), 113.9 (CH),56.0 (CH₃), 51.3 (CH₂), 33.1 (CH₂), 23.5 (CH₂), 21.5 (CH₂), 21.4 (CH₂).ESI-MS m/z=460 ([M+H]⁺). KF=0.08%.

Example 12

Reaction rate of the synthesis of Apixaban from the compound of formula(II) compared with the synthesis of Apixaban from Apixaban ethyl ester.An effect of the invention.

Apixaban can be obtained from compound of formula (II) (as described inExample 9, Example 10 or Example 11) or, according to prior art method,from Apixaban ethyl ester (as described in comparative Example 11), (seealso example 6 of WO2007/0001385).

However, the reaction rate is considerably faster when starting from acompound of formula (II), for example, the compound (II) in which R is—CH₂CH(CH₃)— and CH(CH₃)₂CH₂— (called Propylen glycol ester in FIG. 1and Table 1).

As depicted in FIG. 1, reaction completion (conversion >99%) is reachedwithin 3 h employing the Propylen glycol ester as starting material(triangles in FIG. 1) while it takes at least 6 h, under exactly thesame reaction conditions, from the Apixaban ethyl ester to reach thesame conversion value (circles in FIG. 1).

Moreover, comparing the data collected after 6 hours, the amount ofApixaban is higher when it is prepared from the Propylene glycol ester(99.25% versus 98.95%).

The detailed data are collected in Table 1 for the kinetic study of theconversion of Propylen glycol ester to Apixaban and for the conversionof Apixaban ethyl ester to Apixaban.

TABLE 1 Comparative kinetic study Propylene glycol ester -> Apixabanethyl ester -> Apixaban Apixaban Propylen Apixaban Time (h) glycol esterApixaban ethyl ester Apixaban 0.0 100.0%   0.00% 100.0%  0.00% 1.0 9.46%90.54% 20.86% 61.79% 2.0 1.54% 98.55% — — 3.0 0.83% 99.17% — — 3.5 — — 0.64% 98.25% 6.0 0.75% 99.25%  0.28% 98.95%

Data are expressed as HPLC conversions.

Example 13

HPLC method for the identification and quantification of the compound offormula (II) in which R is —CH(CH₃)₂CH₂—(Isomer A) and —CH₂CH(CH₃)—(Isomer B) which are typical process intermediates and impurities.

As mentioned in Example 10, compound of formula (II), in particular thecompound of formula (II) in which R is —CH₂CH(CH₃)— and —CH(CH₃)₂CH₂—,is also a typical impurity found in the isolated Apixaban productobtained by the process of the invention described in the Examplesabove.

This species could be identified and monitored via the following HPLCmethod:

Chromatographic conditions: Column: XBridge C18 150 × 4.6 mm 3.5 μmTemp. Column: 40° C. Mobile Phase A: H₂O MilliQ/Methanol 90/10 MobilePhase B: Acetonitrile/Methanol 90/10 Time (min) % A % B Gradient: 0 83.516.5 20 5.5 94.5 25 5.5 94.5 Post run: 7 min. Flow: 1.0 mL/min DetectorUV a 252 nm Injection Volume: 5 μL Run Time: 25 min Sample diluent:CH₂Cl₂/EtOH/H₂O 1:5:4

Applying the conditions described above the expected retention times areas indicated below:

Compound RRT Apixaban 1.00 Propylen glycol ester - Isomer B 1.16Propylen glycol ester - Isomer A 1.18 Apixaban ethyl ester 1.467

The amount of the compound of formula (II) into Apixaban is determinedin percent area.

The invention claimed is:
 1. A process for preparing Apixaban form N-1of formula (I):

comprising slurrying Apixaban 1,2-propylen glycol hemisolvate of formula(V):

in a solvent selected from the group consisting of methanol, ethanol,isopropanol, ethyl acetate, a mixture of ethanol and heptane, and amixture of ethanol and water (2:1).
 2. The process of claim 1, whereinthe solvent is selected from the group consisting of ethanol, a mixtureof ethanol and water (2:1), and isopropanol.
 3. The process of claim 1,wherein the slurrying is carried out in ethanol at reflux temperature.4. The process of claim 1, wherein the slurrying is carried out in 40volumes of ethanol.
 5. The process of claim 1, wherein the slurrying iscarried out in a mixture of ethanol and water (2:1) at a temperature of50° C.
 6. The process of claim 1, wherein the slurrying is carried outin 10 volumes of a mixture of ethanol and water (2:1).
 7. The process ofclaim 1, further comprising adding a seed of Apixaban form N-1 offormula (I) to the slurry.
 8. The process of claim 1, wherein theslurrying comprises the steps of: a) adding 40 volumes of ethanol to theApixaban 1,2-propylen glycol hemisolvate of formula (V), b) heating thesuspension of step a) to reflux for 4 hours, and c) cooling thesuspension to temperature of between 20° C. and 25° C., while stirringfor 8 hours.
 9. The process of claim 1, wherein the slurrying comprisesthe steps of: a) adding 10 volumes of a mixture of ethanol and water(2:1) to the Apixaban 1,2-propylen glycol hemisolvate of formula (V) toobtain a slurry, b) seeding the slurry of step a) with Apixaban formN-1, c) heating the seeded slurry to a temperature of 50° C., andstirring the slurry for 2.5 hours, and d) cooling the slurry to roomtemperature, and stirring the slurry for between 2 and 3 hours.
 10. Theprocess of claim 1, wherein the slurrying comprises the steps of: a)adding 10 volumes of ethanol to the Apixaban 1,2-propylen glycolhemisolvate of formula (V) to obtain a slurry, b) seeding the slurry ofstep a) with Apixaban form N-1, and c) stirring the seeded slurry atroom temperature for 4 hours.
 11. The process of claim 1, wherein theApixaban 1,2-propylen glycol hemisolvate of formula (V):

is prepared by reacting ammonia with the compound of formula (III):

wherein R² is Et; in presence of 1,2-propylen glycol.
 12. The process ofclaim 11, wherein the reacting is carried out at a temperature ofbetween 80° C. and 85° C. and at a pressure of 4 bar for 6 hours. 13.The process of claim 1, wherein the Apixaban form N-1 of formula (I) hasthe X-ray powder diffraction pattern with characteristic peaks expressedin 2-Theta values (2θ) at: 8.4, 10.0, 10.5, 11.1, 12.3, 12.8, 13.9,15.1, 16.2, 16.9, 18.4, 18.8, 19.6, 21.1, 21.5, 22.0, 22.2, 23.6, 24.0,24.7, 25.3, 26.2, 26.9, 27.7, 28.0, 28.6, 29.2, 29.9, 30.6, 31.9, 32.6,35.1, each peak ±0.1.
 14. The process of claim 1, wherein the Apixabanform N-1 of formula (I) has a melting point of 235.68° C. as measured byDSC.
 15. The process of claim 1, wherein the Apixaban 1,2-propylenglycol hemisolvate of formula (V) has the X-ray powder diffractionpattern with characteristic peaks expressed in 2-Theta values (2θ) at:6.6, 7.6, 8.1, 9.9, 11.7, 12.7, 13.7, 14.5, 15.1, 15.6, 16.3, 16.9,17.2, 17.9, 18.2, 19.5, 20.0, 20.5, 20.8, 21.4, 22.8, 23.8, 24.8, 25.5,29.0, 31.2, 33.0, each peak ±0.1.
 16. The process of claim 1, whereinthe Apixaban 1,2-propylen glycol hemisolvate of formula (V) has amelting point of 195° C. as measured by DSC.
 17. A process for preparingApixaban form N-1 of formula (I):

comprising crystallizing Apixaban 1,2-propylen glycol hemisolvate offormula (V):

from methanol or ethanol to give Apixaban form N-1.
 18. The process ofclaim 17, wherein the crystallization is carried out in either 36volumes of methanol or 60 volumes of ethanol.
 19. The process of claim17, further comprising adding a seed of Apixaban form N-1 to theApixaban 1,2-propylen glycol hemisolvate of formula (V) in methanol orethanol.
 20. The process of claim 17, wherein the crystallization iscarried out from reflux temperature to room temperature.